Determining the gas-phase structures of α-helical peptides from shape, microsolvation, and intramolecular distance data

Mass spectrometry is a powerful technique for the structural and functional characterization of biomolecules. However, it remains challenging to accurately gauge the gas-phase structure of biomolecular ions and assess to what extent native-like structures are maintained. Here we propose a synergistic approach which utilizes Förster resonance energy transfer and two types of ion mobility spectrometry (i.e., traveling wave and differential) to provide multiple constraints (i.e., shape and intramolecular distance) for structure-refinement of gas-phase ions. We add microsolvation calculations to assess the interaction sites and energies between the biomolecular ions and gaseous additives. This combined strategy is employed to distinguish conformers and understand the gas-phase structures of two isomeric α-helical peptides that might differ in helicity. Our work allows more stringent structural characterization of biologically relevant molecules (e.g., peptide drugs) and large biomolecular ions than using only a single structural methodology in the gas phase.

cR6G-tmP1, and (e-g) rh110-tmP2, rh110-tmP3, and rh110-tmP4. The distance between the two cysteines in P1 and P2 was optimized to fit for the measurements of large biomolecular backbone distances by traditional FRET.

Supplementary Note 2. DMS, IMS and tmFRET methods
Transition metal ion FRET (tmFRET) is a recent extension of FRET experiment in the gas phase, which enable the measurement of shorter biomolecular backbone distances (10−40 Å) compared to traditional FRET. 1 In this work, four polyalanine-based peptides (tmP1-4) were labeled with rhodamine110 (rh110) or carboxyrhodamine 6g (cR6G) as donor fluorophore. The acceptor dye was replaced with a transition metal ion (i.e., Cu 2+ ), which bind noncovalently to a His-X3-His motif. With a simplified lifetime measurement of donor dye signal reduction, tmFRET preserves high distance sensitivity and could serve as a molecular ruler in the gas phase. tmFRET also facilitates small Förster distance (R0) values compared to traditional FRET. Therefore, tmFRET is more sensitive to conformational changes of peptides and proteins. In the tmFRET model peptides, Cu 2+ was found to be bound with the histidine close to the Nterminal, which was confirmed by molecular dynamics (MD) simulation and collision-induced dissociation (CID). 1 Surface-induced dissociation (SID) experiments, which generally produce compact and native-like subcomplex fragments of multiply charged proteins, was also conducted for the tmFRET model peptides. 2 Compared with CID, SID provides better energy transfer in theory.
Therefore, SID avoids a slow heating effect of ions in CID and results in a less Cu 2+ rearrangement during fragmentation process. Overall, SID results also confirmed similar binding site for Cu 2+

Supplementary Note 2.1. Lifetime measurements and distance estimation from FRET efficiency (E)
The estimated rDA from the FRET efficiencies of cR6G-tmP1 (from Supplementary Figures 2a   and 3) in the gas phase are discussed in the manuscript. The distance increasement indicated an expanded helical structure with increasing charge state, which is likely due to Coulomb repulsion.
However, previous MD simulations of rh110-tmP2, -tmP3, and -tmP4 using AMBER ff14SB force field showed a broad radial distribution of donor-acceptor distances. 1 This indicated the possibility of multiple conformations in specific charge states. Multiple conformations would lead to convoluted fluorescence decay curve, in which single-exponential fit is usually insufficient.
Moreover, fluorescence decay curves acquired in the gas phase are typically in a limited quality as the low ion density and small fluorescence collection angle. Therefore, the resulting fluorescence decay curve is challenging to be analyzed even with a multi-exponential fits. Determination of distances based on FRET (or tmFRET) could be more accurate if multiple species are separated or in combination with IM-MS. Therefore, we hypothesized that an online coupling with DMS separation to fluorescence spectroscopy could potentially disentangle the complexity of data set. [M+Cu+H] 3+ ions of cR6G-tmP1 with λex = 460 nm, P = 10mW. [M+Cu+3H] 5+ signal was acquired with 3*3600 s of fluorescence collection, another two ions were acquired with 3*600 s of fluorescence collection. The fluorescence decay curves were fitted with a single-exponential modified Gaussian function (red) or double-exponential fit (blue). The arrow shows the tmFRET pathway between the position of labeled dye and where Cu 2+ is likely to be bound.  to "native" state in native MS, and peptide ions are likely to retain their folded structure. As the charge state increases, peptide ions quickly unfold (likely 3+ charge state) and stabilize into one or two conformations (4+ and 5+ charge states). As shown in Supplementary Figure 5, slightly different trends of CCS increment from 2+ to 4+ (or 5+ for cR6G-tmP1) upon Cu 2+ bound were observed for these peptides. In general, the trend of cR6G-tmP1 protonated species increase linearly, while rh110-tmP2, rh110-tmP3, and rh110-tmP4 of dramatically from 2+ to 3+ charge state, then flattened from 3+ to 4+. Specifically, the [M+2H] 2+ ion of cR6G-tmP1 has the CCS compared   Figure 5. Corrected N2 CCS values for the four tmFRET model peptide ions. Only the major conformer in the ion mobility spectra for the Cu-free species (blue) and Cu-bound complexes (green) ions are plotted. Binding with Cu 2+ induces alternation of peptide structure in the gas phase.

Supplementary Note 2.3. DMS experiments
DMS separations of four tmFRET model peptides were then conducted and results are presented in Supplementary Figures 6-14. When no gas modifier was applied, poor separation was achieved. Isopropanol (IPA), serving as a common gas modifier, was then doped into the carrier gas. Peptide ions were shifted from negative compensation voltage (CV) to positive CV due to dynamic clustering/declustering mechanisms. 4,5 As shown in Supplementary Figure 6   of an ion is a global shape (or size) parameter and may not limit to only one conformation. FRET or tmFRET, which provides intramolecular distance, is potentially capable to distinguish ions of different conformation with similar CCS. Therefore, we hypothesized that the computational simulation can be guided for better determination of the gas-phase ion structures.
DMS separation, which is proved to be a complementary technique to IM-MS and FRET or tmFRET, represents the cluster formation properties between a charged ion and (one or several) neutral gas modifier molecules. Even though the ion-molecular interaction varies between analytes, the binding energy and possible solvation sites available for binding of gas modifier molecules could be calculated for each candidate conformer. Therefore, DMS results were incorporated to the selection of candidate structures in the gas phase, together with IM-MS and FRET-based methods.  Figure 19. Gas  Triwave, which is after the TWIM separation. TWIM separation was assumed not to be affected.

Supplementary Note 3. Differential ion mobility spectrometry (DMS) instrumentation and experiments
Conformer 1 and 3 also showed high ratio of y ions, while conformer 2 and 4 showed high ratio of b ions (Supplementary Tables 5-8). The difference is likely driven by a more favorable charging in the basic side chains of amino acids further away from the labelling dye (likely singly charge) due to Coulomb repulsion. The relatively low ratio of y-Atto 532 ions for conformers 1 and 3, and Atto 532 ionsb ions for conformers 2 and 4 are likely due to partially overlapped peaks in the CCS distributions.

Supplementary Note 5.2. Fluorescence spectroscopic experiments in the solution phase
Solution-phase FRET experiments of cR6G-P1-QSY7 and cR6G-P2-QSY7 were conducted in an in-plume setup by replacing the nano-electrospray tip with a cuvette. 8 The results showed two conformations from the double-exponential fit (Supplementary Figure 26 and Supplementary Table 9).
Both cR6G-P1-QSY7 conformers exhibited a slightly lower lifetime as well as a higher ratio of molecules adopting compact conformations (58%) compared to cR6G-P2-QSY7 (40%).    The results obtained for a double-exponential fit of the doubly labelled peptides are listed in Supplementary